OBJECTIVE Respiratory infections with multidrug-resistant (MDR) bacteria are life-threatening in patients with cystic fibrosis (CF). Squalamine and aminosterol derivatives (ASDs) have previously demonstrated interesting antibacterial activity against bacterial reference strains. This study investigated for the first time their activity against MDR clinical isolates recovered from the sputa of CF patients. METHODS Antibacterial activity of squalamine and two ASDs (1 and 2) was evaluated against 135 MDR gram-negative and gram-positive bacteria using the broth microdilution method for MIC determination. RESULTS For gram-negative bacteria, MICs ranged from 2 to 128 mg/L. Resistance to colistin and mucoidity were significantly associated with higher MICs of squalamine and ASDs 1 and 2. Tested compounds were active against various gram-positive bacteria with MIC values varying from 0.5 to 8 mg/L, with the exception of two capsulated isolates of Streptococcus pneumoniae demonstrating MICs of 32 mg/L. CONCLUSIONS In this study, we present new findings concerning the antibacterial potential of ASDs against MDR bacteria. Colistin-resistant, mucoid and capsulated bacteria were found to exhibit decreased susceptibility to ASDs indicating that these compounds might share some mechanistic aspects with polymyxins towards gram-negative bacteria. However, ASDs were remarkably active against gram-positive species suggesting different mechanisms of action towards gram-positive and gram-negative bacteria. As tested ASDs exhibited elevated MICs in some cases, we believe that these compounds may be developed to be locally administrated as aerosols rather than via systemic administration routes. Further work is warranted to evaluate their in vivo efficacy in aerosol formulations using a lung-infected animal model.